Antibody formulation

ABSTRACT

Anti-CD40 antibodies are formulated as lyophilisate or liquid formulation. The lyophilisates can be reconstituted to give a solution with a high concentration of the antibody active ingredient for delivery to a patient without high levels of antibody aggregation. The lyophilisate can be reconstituted with an aqueous reconstituent to provide an aqueous composition in which the antibody has a concentration of at least 50 mg/ml. The lyophilisate or aqueous pharmaceutical composition may include one or more of a sugar, a buffering agent, a surfactant, and/or a free amino acid.

TECHNICAL FIELD

The present invention relates to a pharmaceutical formulation of anantibody against CD40, a process for the preparation thereof and uses ofthe formulation.

BACKGROUND

Despite the availability of several immunosuppressive treatments forautoimmune diseases, there remains a large unmet need for moreefficacious and safer drugs in a large fraction of the patientpopulation. For example, despite the reported efficacy of B celldepleting/inhibiting therapies like Rituximab and Belimumab inrheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome,and multiple sclerosis, these therapies are only effective in a portionof diseased individuals, and with Rituximab, with an accompanying riskof progressive multifocal leukoencephalopathy. Further, multiple otherleukocyte cell types are often involved in the pathology of theseautoimmune diseases such as macrophages, dendritic cells and T cells,therefore therapeutic intervention targeting additional cell types orkey immunological pathways that would inhibit their function couldprovide benefit. Given the multiple immunologically relevant roles ofCD40-CD154 in the activation and function or these cell types, it islikely that an anti-CD40 antibody would confer therapeutic benefit topatients suffering autoimmune diseases outlined above beyond thatcurrently provided by current therapies. Further, the central role forCD40-CD154 interactions in intestinal inflammatory disorders such asCrohn's disease and ulcerative colitis, and mechanistic links of theCD40 pathway to pathology in more rare disorders such as autoimmunevasculitis, pemphigus vulgaris, and idiopathic thrombocytopenic purpura(ITP) also highlights the potential of anti-CD40 antibodies in theseindications.

The currently available immunosuppressants used after solid organtransplantation provide excellent short-tem efficacy. Acute rejectionswithin the de novo period are observed in 5%-20% of the recipients(depending on organ, patient population, and regimen) and the proportionof grafts lost to acute rejection within the de novo period is below 5%for any setting. Currently the key unmet need is the tolerability ofimmunosuppression with patient and graft survival in the long term.After renal transplant, 33% patients die and/or lose their graft within5 years; the average age of death of transplant recipient is 58 years.Calcineurin inhibitors (CNI) remain the mainstay of immunosuppressivetherapy for the vast majority of transplant patients. Whilenephrotoxicity and cardiovascular morbidity associated with CNIs is oneof the drivers of chronic allograft nephropathy as well as patient deathwith a functioning graft, alternative primary immunosuppression have notbeen able to replace CNIs. Overall, there is still room for improvementin long-term transplant immunosuppression. B-cell mediated immunologicaldamage of transplanted kidneys may contribute to poor long-term outcomesand the need for new agents to target B-cell rejection is increasinglyrecognised by the medical community.

Antibodies against CD40 are known in the art. Chir12.12 is a fullyhumanised, non-agonist anti-CD40 antibody (IgG1, kappa) that blocksCD154 (also known as CD40 ligand; CD40L)-mediated leukocyte activationand can mediate antibody-dependent cellular cytotoxicity (ADCC) of humanleukocytes and B cell lymphomas in vitro (see WO 2006/073443). WO2005/044306 describes anti-CD40 antagonist antibodies, including

Chir12.12 for use in particular in the treatment of autoimmune andinflammatory disorders. Further Chir12.12 is effective in delayingkidney allograft rejection when dosed as a monotherapy in Macacafascicularis (Cynomolgus monkeys) [Li et al. (2008) Transplantation; 86(1):10-15]. However, Chir12.12 can also mediate depletion of peripheralB cells in non human primates (NHPs).

Anti-CD40 mAbs with silenced ADCC activity are predicted to have animproved safety profile relative to the parental anti-CD40 antibodies,and in particular may be more suitable for non-oncologic indications,such as autoimmune diseases and use in a transplant setting. Theapplicant has developed three silent anti-CD40 antibodies based on theChir12.12 antibody. These antibodies, hereinafter designated mAb1, mAb2and mAb3 are characterised by certain amino acid mutations in the Fcregion which silence ADCC activity.

mAb1 comprises an N297A mutation in the antibody heavy chain amino acidsequence. The antibody comprises the heavy chain CDR1, CDR2 and CDR3 ofSEQ ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2 and CDR3of SEQ ID NOs: 6, 7 and 8, respectively. mAb1 comprises a V_(H) domainwith the amino acid sequence of SEQ ID NO: 1 and a V_(L) domain with theamino acid sequence of SEQ ID NO: 2. mAb1 comprises the full lengthheavy chain amino acid sequence of SEQ ID NO: 9 and the full lengthlight chain amino acid sequence of SEQ ID NO: 10.

mAb2 comprises a D265A mutation in the antibody heavy chain amino acidsequence. The antibody comprises the heavy chain CDR1, CDR2 and CDR3 ofSEQ ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2 and CDR3of SEQ ID NOs: 6, 7 and 8, respectively. mAb2 comprises a V_(H) domainwith the amino acid sequence of SEQ ID NO: 1 and a V_(L) domain with theamino acid sequence of SEQ ID NO: 2. mAb2 comprises the full lengthheavy chain amino acid sequence of SEQ ID NO: 13 and the full lengthlight chain amino acid sequence of SEQ ID NO: 14.

mAb3 comprises a L234A, L235A mutation (LALA) in the antibody heavychain amino acid sequence. The antibody comprises the heavy chain CDR1,CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and light chainCDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8, respectively. mAb3comprises a V_(H) domain with the amino acid sequence of SEQ ID NO: 1and a V_(L) domain with the amino acid sequence of SEQ ID NO: 2. mAb3comprises the full length heavy chain amino acid sequence of SEQ ID NO:17 and the full length light chain amino acid sequence of SEQ ID NO: 18.

Formulations with high concentration of antibody may have short shelflives and the formulated antibodies may lose biological activityresulting from chemical and physical instabilities during the storage.Among those, aggregation, deamidation and oxidation are known to be themost common causes of antibody degradation. In particular, aggregationcan potentially lead to increased immune response in patients, leadingto safety concerns. Thus it must be minimised or prevented.

It is an object of the invention to provide further and improvedformulations of anti-CD40 antibodies, and in particular formulationswith high concentration of anti-CD40 antibodies and low levels ofantibody aggregation.

DISCLOSURE OF THE INVENTION

Therapeutic antibodies are typically formulated either in aqueous formready for parenteral administration or as lyophilisates forreconstitution with a suitable diluent prior to administration.According to the invention, an anti-CD40 antibody may be formulatedeither as a lyophilisate, or as an aqueous composition, for example inpre-filled syringes. Suitable formulation can provide an aqueouspharmaceutical composition or a lyophilisate which can be reconstitutedto give a solution with a high concentration of the antibody activeingredient and a low level of antibody aggregation for delivery to apatient. High concentrations of antibody are useful as they reduce theamount of material which must be delivered to a patient. Reduced dosingvolumes minimise the time taken to deliver a fixed dose to the patient.The aqueous compositions of the invention with high concentration ofanti-CD40 antibodies are particularly suitable for subcutaneousadministration.

Thus the invention provides an aqueous pharmaceutical composition,suitable for parenteral administration in a subject, e.g., forsubcutaneous administration, comprising an anti-CD40 antibody.

The following specific embodiments of the invention are described asnumbered hereafter:

-   1. An aqueous pharmaceutical composition, suitable for subcutaneous    administration in a subject, comprising an anti-CD40 antibody in    which the antibody has a concentration of at least 50 mg/ml, and    wherein said anti-CD40 antibody includes: (i) one or more heavy    chain CDRs selected from the group consisting of SEQ ID NOs: 3, 4    and 5; and/or (ii) one or more light chain CDRs selected from the    group consisting of SEQ ID NOs: 6, 7 and 8.-   2. The aqueous pharmaceutical composition, suitable for subcutaneous    administration in a subject, according to Embodiment 1, wherein said    anti-CD40 antibody includes heavy chain CDR1, CDR2 and CDR3 of SEQ    ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2 and CDR3    of SEQ ID NOs: 6, 7 and 8.-   3. The aqueous pharmaceutical composition of Embodiment 1 or 2,    wherein the anti-CD40 antibody comprises a V_(H) domain with amino    acid SEQ ID NO: 1 and a V_(L) domain with amino acid SEQ ID NO: 2.-   4. The aqueous pharmaceutical composition of Embodiment 1, 2 or 3,    wherein the anti-CD40 antibody comprises a heavy chain region of SEQ    ID NO: 9 and a light chain region of SEQ ID NO: 10, or a heavy chain    region of SEQ ID NO: 13 and a light chain region of SEQ ID NO: 14,    or a heavy chain region of SEQ ID NO: 17 and a light chain region of    SEQ ID NO: 18.-   5. The aqueous pharmaceutical composition of any one of Embodiments    1 to 4, wherein less than 5% of the anti-CD40 antibody is aggregated    or degraded.-   6. The aqueous pharmaceutical composition of any one of Embodiments    1 to 5, comprising one or more of the following components selected    among the group consisting of: a stabiliser, a buffering agent; and    a surfactant.-   7. The aqueous pharmaceutical composition of Embodiment 6, wherein    the stabiliser is a sugar.-   8. The aqueous pharmaceutical composition of Embodiment 6 or 7,    comprising: a sugar, a buffering agent, and a surfactant.-   9. The aqueous pharmaceutical composition of Embodiment 6 or 7,    further comprising a free amino acid.-   10. The aqueous pharmaceutical composition of Embodiment 7 to 9,    comprising sucrose as a sugar.-   11. The aqueous pharmaceutical composition of Embodiment 10,    comprising 200-300 mM sucrose.-   12. The aqueous pharmaceutical composition of Embodiments 6-11,    comprising a histidine buffer as the buffering agent.-   13. The aqueous pharmaceutical composition of Embodiment 12,    comprising 25-35 mM histidine buffer.-   14. The aqueous pharmaceutical composition of Embodiments 6 to 13,    comprising polysorbate 20 as a surfactant.-   15. The aqueous pharmaceutical composition of Embodiment 14,    comprising 0.01 to 0.2% polysorbate 20.-   16. The aqueous pharmaceutical composition of Embodiment 9, further    comprising arginine and/or methionine as free amino acid.-   17. The aqueous pharmaceutical composition of Embodiment 16,    comprising 40-80 mM arginine.-   18. The aqueous pharmaceutical composition of any preceding    Embodiment, comprising sucrose, a histidine buffer, polysorbate 20    and arginine.-   19. The aqueous pharmaceutical composition of any preceding    Embodiment, comprising sucrose, a histidine buffer, polysorbate 20    and methionine.-   20. The aqueous pharmaceutical composition of any preceding    Embodiment, comprising sucrose, a histidine buffer, polysorbate 20,    arginine and methionine.-   21. A lyophilisate suitable for preparing the aqueous pharmaceutical    composition of any preceding Embodiments.-   22. A lyophilisate according to Embodiment 21, comprising sucrose, a    histidine buffer, and polysorbate 20.-   23. A method for preparing a lyophilisate, comprising the steps    of: (i) preparing an aqueous solution comprising an anti-CD40    antibody, a sugar, a buffering agent, a surfactant and optionally a    free amino acid; and (ii) lyophilising the aqueous solution.-   24. A delivery device including the aqueous pharmaceutical    composition of any one of Embodiments 1-20.-   25. A pre-filled syringe including the aqueous pharmaceutical    composition of any one of Embodiments 1-20.-   26. A method for delivering an anti-CD40 monoclonal antibody to a    mammal, comprising a step of administering to the patient a    pharmaceutical composition of any one of Embodiments 1-20.-   27. The composition of any one of Embodiments 1-20, for use in    treating a disease or disorder that is mediated by CD40.-   28. The composition of Embodiment 27, for the treatment of    autoimmune diseases.-   29. The composition of Embodiment 28, for the treatment of    rheumatoid arthritis, systemic lupus erythematosus, or Pemphigus    vulgaris.-   30. The aqueous pharmaceutical composition of any one of Embodiments    1-20 in which the antibody has a concentration of at least at least    50 mg/ml, at least 100 mg/ml, at least 150 mg/ml, at least 200    mg/ml, at least 250 mg/ml, or at least 300 mg/ml.

The invention also provides an aqueous pharmaceutical compositioncomprising: an anti-CD40 monoclonal antibody as described above, forexample mAb1, mAb2 or mAb3, especially mAb1; a stabiliser; a bufferingagent; and a surfactant. The composition preferably also includes a freeamino acid.

The invention also provides a lyophilisate comprising: an anti-CD40monoclonal antibody as described above, for example mAb1, mAb2 or mAb3,especially mAb1; a sugar; a buffering agent; and a surfactant. Thelyophilisate preferably also includes a free amino acid.

The invention also provides a lyophilisate comprising an anti-CD40monoclonal antibody as described above, for example mAb1, mAb2 or mAb3,especially mAb1; wherein the lyophilisate can be reconstituted with anaqueous reconstituent to provide an aqueous composition in which theantibody has a concentration of at least 50 mg/ml, 100 mg/ml, 150 mg/ml,200 mg/ml, 250 mg/ml, or 300 mg/ml, after reconstitution in an aqueoussolution.

The invention also provides an aqueous pharmaceutical compositioncomprising high concentration of an anti-CD40 monoclonal antibody asdescribed above, for example mAb1, mAb2 or mAb3, especially mAb1;wherein less than 5%, 4%, 3%, 2% or 1% of the anti-CD40 antibody isaggregated or degraded.

The invention also provides a process for preparing a lyophilisate,comprising steps of: (i) preparing an aqueous solution comprising ananti-CD40 monoclonal antibody, a sugar, a buffering agent, a surfactant,and optionally a free amino acid; and (ii) lyophilising the aqueoussolution.

The invention also provides a process for preparing a pharmaceuticalcomposition, comprising a step of mixing a lyophilisate with an aqueousreconstituent, wherein the lyophilisate comprises an anti-CD40monoclonal antibody, a sugar, a buffering agent, a surfactant, andoptionally a free amino acid.

More specifically the invention provides a lyophilised formulationprepared by lyophilising an aqueous formulation having a pH of 5.0-7.0and comprising

-   (i) an anti-CD40 antibody wherein the antibody has a concentration    of 20-150 mg/ml, and wherein said anti-CD40 antibody includes heavy    chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and    light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8,-   (ii) a stabiliser,-   (iii) a buffering agent,-   (iv) a surfactant, and optionally-   (v) an amino acid.-   In one embodiment said formulation is prepared from an aqueous    formulation having a pH of 5.0-7.0 and comprising-   (i) an anti-CD40 antibody wherein the antibody has a concentration    of 20-150 mg/ml, and wherein said anti-CD40 antibody includes heavy    chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and    light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8,-   (ii) sucrose or trehalose as a stabiliser,-   (iii) histidine as a buffering agent,-   (iv) polysorbate 20 as a surfactant, and optionally-   (v) an amino acid selected from arginine, methionine and glycine.-   In one embodiment said lyophilised formulation is prepared from an    aqueous formulation having a pH of 5.0-7.0 and comprising-   (i) an anti-CD40 antibody wherein the antibody has a concentration    of 20-150 mg/ml, and wherein said anti-CD40 antibody includes heavy    chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and    light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8,-   (ii) 3-300 mM sucrose or trehalose as a stabiliser,-   (iii) 1-60 mM histidine as a buffering agent,-   (iv) up to 0.2% polysorbate 20 as a surfactant, and optionally-   (v) 2-80 mM arginine, methionine or glycine.-   In one embodiment said lyophilised formulation is prepared from an    aqueous formulation having a pH of 6.0 and comprising-   (i) an anti-CD40 antibody wherein the antibody has a concentration    of 50 mg/ml, and wherein said anti-CD40 antibody includes heavy    chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and    light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8,-   (ii) 90 mM sucrose as a stabiliser,-   (iii) 10 mM histidine as a buffering agent,-   (iv) 0.02% polysorbate 20 as a surfactant.-   In one embodiment said lyophilised formulation is prepared from an    aqueous formulation having a pH of 6.0 and comprising-   (i) an anti-CD40 antibody wherein the antibody has a concentration    of 50 mg/ml, and wherein said anti-CD40 antibody includes heavy    chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and    light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8,-   (ii) 90 mM sucrose as a stabiliser,-   (iii) 10 mM histidine as a buffering agent,-   (iv) 0.02% polysorbate 20 as a surfactant, and-   (v) 17 mM arginine.-   In one embodiment said lyophilised formulation is prepared from an    aqueous formulation having a pH of 6.0 and comprising-   (i) an anti-CD40 antibody wherein the antibody has a concentration    of 150 mg/ml, and wherein said anti-CD40 antibody includes heavy    chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and    light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8,-   (ii) 270 mM sucrose as a stabiliser,-   (iii) 30 mM histidine as a buffering agent, and-   (iv) 0.06% polysorbate 20 as a surfactant.-   The invention also provides an aqueous pharmaceutical composition    obtained by reconstituting a lyophilised formulation as described    above, wherein the reconstitution factor is between 1:0.5 to 1:6.

In one embodiment the reconstitution factor is 1:3.

-   The invention also provides an aqueous pharmaceutical composition    having a pH of 5.0 to 7.0 comprising    -   (i) an anti-CD40 antibody wherein the antibody has a        concentration of at least 50 mg/ml, and wherein said anti-CD40        antibody includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs        3, 4 and 5 respectively, and light chain CDR1, CDR2 and CDR3 of        SEQ ID NOs: 6, 7 and 8,    -   (ii) a stabiliser,    -   (iii) a buffering agent,    -   (iv) a surfactant, and optionally    -   (v) an amino acid.-   In one embodiment the aqueous pharmaceutical composition having a pH    of 5.0 to 7.0 comprises    -   (i) an anti-CD40 antibody wherein the antibody has a        concentration of at least 50 mg/ml, and wherein said anti-CD40        antibody includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs        3, 4 and 5 respectively, and light chain CDR1, CDR2 and CDR3 of        SEQ ID NOs: 6, 7 and 8,    -   (ii) sucrose or trehalose as a stabiliser,    -   (iii) histidine as a buffering agent,    -   (iv) polysorbate 20 as a surfactant, and optionally    -   (v) an amino acid selected from arginine, methionine or glycine.-   In one embodiment the aqueous pharmaceutical composition having a pH    of 5.0 to 7.0 comprises    -   (i) an anti-CD40 antibody wherein the antibody has a        concentration of at least 50 mg/ml, and wherein said anti-CD40        antibody includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs        3, 4 and 5 respectively, and light chain CDR1, CDR2 and CDR3 of        SEQ ID NOs: 6, 7 and 8,    -   (ii) 200-300 mM sucrose as a stabiliser,    -   (iii) 25-35 mM histidine as a buffering agent,    -   (iv) up to 0.2% polysorbate 20 as a surfactant, and optionally    -   (v) 10-80 mM arginine, methionine or glycine.-   In one embodiment the aqueous pharmaceutical composition has a pH of    6.0 and comprises-   (i) an anti-CD40 antibody wherein the antibody has a concentration    of at least 50 mg/ml, and wherein said anti-CD40 antibody includes    heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5    respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6,    7 and 8,-   (ii) 270 mM sucrose as a stabiliser,-   (iii) 30 mM histidine as a buffering agent, and-   (iv) 0.06% polysorbate 20 as a surfactant.-   In one embodiment the aqueous pharmaceutical composition has a pH of    6.0 and comprises-   (i) an anti-CD40 antibody wherein the antibody has a concentration    of at least 50 mg/ml, and wherein said anti-CD40 antibody includes    heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5    respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6,    7 and 8,-   (ii) 270 mM sucrose as a stabiliser,-   (iii) 30 mM histidine as a buffering agent,-   (iv) 0.06% polysorbate 20 as a surfactant, and-   (v) 51 mM arginine.-   In one embodiment the aqueous pharmaceutical composition of the    invention has an anti-CD40 antibody concentration of 150 mg/ml.-   In one embodiment the lyophilised formulation or the aqueous    pharmaceutical composition of the invention comprises a VH domain    having the amino acid sequence of SEQ ID NO: 1 and a VL domain    having the amino acid sequence of SEQ ID NO: 2.-   In one embodiment the lyophilised formulation or the aqueous    pharmaceutical composition of the invention comprises an anti-CD40    antibody comprising a heavy chain region of SEQ ID NO: 9 and a light    chain region of SEQ ID NO: 10.-   In one embodiment the lyophilised formulation or the aqueous    pharmaceutical composition of the invention comprises the anti-CD40    antibody Chir12.12 having an N297A mutation.-   In one embodiment the lyophilised formulation or the aqueous    pharmaceutical composition of the invention comprises an anti-CD40    antibody comprising a heavy chain region of SEQ ID NO: 13 and a    light chain region of SEQ ID NO: 14.-   In one embodiment the lyophilised formulation or the aqueous    pharmaceutical composition of the invention comprises the anti-CD40    antibody Chir12.12 having a D265A mutation.-   In one embodiment the lyophilised formulation or the aqueous    pharmaceutical composition of the invention comprises an anti-CD40    antibody comprising a heavy chain region of SEQ ID NO: 17 and a    light chain region of SEQ ID NO: 18.-   In one embodiment the lyophilised formulation or the aqueous    pharmaceutical composition of the invention comprises the anti-CD40    antibody Chir12.12 having an L234A L235A mutation.-   The invention also comprises a delivery device including the aqueous    pharmaceutical composition of the invention.-   The invention also comprises a pre-filled syringe including the    aqueous pharmaceutical composition of the invention.-   The invention also comprises a method for delivering an anti-CD40    antibody to a mammal, comprising a step of administering to the    patient an aqueous pharmaceutical composition of the invention.-   The invention also comprises a lyophilised formulation or an aqueous    pharmaceutical composition according to the invention for use in    treating a disease or disorder that is mediated by CD40.-   The invention also comprises a lyophilised formulation or aqueous    pharmaceutical composition according to the invention for use in the    treatment of autoimmune diseases.-   The invention also provides a lyophilised formulation or aqueous    pharmaceutical composition according to the invention Multiple    Sclerosis, Systemic Lupus Erythematosus, Sjögren's syndrome,    Rheumatoid Arthritis, transplant rejection; graft-versus-host    disease, Pemphigus vulgaris.    Aqueous Pharmaceutical Compositions with High Concentration of    Anti-CD40 Antibodies

The invention relies, at least partly, in the formulation properties ofantibodies such as mAb1, which retain remarkable stability and bioactiveproperties when formulated in a high concentration either as a liquid(aqueous) or lyophilisate composition.

As used herein, an “aqueous” pharmaceutical composition is a compositionsuitable for pharmaceutical use, wherein the aqueous carrier isdistilled water. A composition suitable for pharmaceutical use may besterile, homogeneous and/or isotonic. Aqueous pharmaceuticalcompositions may be prepared either directly in an aqueous form, forexample in pre-filled syringe ready for use (the “liquid formulations”)or as lyophilisate to be reconstituted shortly before use. As usedherein, the term “aqueous pharmaceutical composition” refers to theliquid formulation or reconstituted lyophilised formulation. In certainembodiments, the aqueous pharmaceutical compositions of the inventionare suitable for parenteral administration to a human subject. In aspecific embodiment, the aqueous pharmaceutical compositions of theinvention are suitable for subcutaneous administration.

As used herein, the phrase “parenteral administration” means mode ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

The use of antibodies as the active ingredient of pharmaceuticals is nowwidespread, including the products HERCEPTIN™ (trastuzumab), RITUXAN™(rituximab), SYNAGIS™ (palivizumab), etc. Techniques for purification oftherapeutic antibodies to a pharmaceutical grade are well known in theart.

The composition will usually be non-pyrogenic e.g. containing <1 EU(endotoxin unit, a standard measure) per dose, and preferably <0.1 EUper dose. The composition is preferably gluten-free.

In specific embodiments, the aqueous pharmaceutical compositions of theinvention exhibit low to undetectable levels of antibody aggregation ordegradation, with very little to no loss of the biological activitiesduring manufacture, preparation, transportation and long periods ofstorage, the concentration of the anti-CD40 antibody being at leastabout 50 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300mg/ml.

In one aspect, the invention relates to an aqueous pharmaceuticalcomposition with high concentration of anti-CD40 antibodies.

It is known in the art that such high concentration aqueouspharmaceutical compositions can be diluted prior to injection, forexample, if lower antibody concentrations are required for specifictherapeutic interventions or when treating patients of lower body weightincluding children. Suitable concentrations can be 25 mg/ml or 10 mg/ml.Alternatively, the original formulation may be produced with such alower concentration.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i. e., “antigen-binding portion”) orsingle chains thereof. A naturally occurring “antibody” is aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain is comprisedof a heavy chain variable region (abbreviated herein as V_(H)) and aheavy chain constant region. The heavy chain constant region iscomprised of three or four domains, depending on the isotype, C_(H)1,C_(H)2, C_(H)3 and C_(H)4. Each light chain is comprised of a lightchain variable region (abbreviated herein as V_(L)) and a light chainconstant region. The light chain constant region is comprised of onedomain, C_(L). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (Clq) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to full length or one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., a portion of CD40). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains; a F(ab)₂ fragment, a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the V_(H) and C_(H)1 domains; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a V_(H) domain; and an isolated complementarity determiningregion (CDR).

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding region” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities, e.g., an isolated antibody that specifically binds humanCD40 is substantially free of antibodies that specifically bind antigensother than CD40. An isolated antibody that specifically binds CD40 may,however, have cross-reactivity to other antigens, such as CD40 moleculesfrom other species. Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, includes antibodies havingvariable regions in which both the framework and CDR regions are derivedfrom sequences of human origin. Furthermore, if the antibody contains aconstant region, the constant region also is derived from such humansequences, e.g., human germline sequences, or mutated versions of humangermline sequences or antibody containing consensus framework sequencesderived from human framework sequences analysis, for example, asdescribed in Knappik, et al. (2000. J Mol Biol 296, 57-86).

The structures and locations of immunoglobulin variable domains, e.g.,CDRs, may be defined using well known numbering schemes, e.g., the Kabatnumbering scheme, the Chothia numbering scheme, a combination of Kabatand Chothia (AbM), etc. (see, e.g., Sequences of Proteins ofImmunological Interest, U.S. Department of Health and Human Services(1991), eds. Kabat et al.; Al Lazikani et al. (1997) J. Mol. Bio.273:927 948). Throughout this specification, the complementaritydetermining region (“CDR”) is defined according to the Kabat definitionwith the exception of CDRH1 which is the stretch of amino acids definedby a combination of both Kabat and Chothia definitions for this CDR.

The human antibodies of the invention may include amino acid residuesnot encoded by human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM, IgA,IgD, IgE and IgG such as IgG1, IgG2, IgG3 or IgG4) that is provided bythe heavy chain constant region genes.

The phrases “an antibody recognising an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen”.

As used herein, an antibody that “specifically binds to CD40polypeptide” or an “anti-CD40 antibody” refers to an antibody that bindsto human CD40 polypeptide of SEQ ID NO: 21 with a K_(D) of 100 nM orless, 10 nM or less, 1 nM or less. An antibody that “cross-reacts withan antigen other than CD40” refers to an antibody that binds thatantigen with a K_(D) of 0.5×10⁻⁸ M or less, 5×10⁻⁹ M or less, or 2×10⁻⁹M or less. An antibody that “does not cross-react with a particularantigen” is intended to refer to an antibody that binds to that antigen,with a K_(D) of 1.5×10⁻⁸ M or greater, or a K_(D) of 5-10×10⁻⁸ M or1×10⁻⁷ M or greater. In certain embodiments, such antibodies that do notcross-react with the antigen exhibit essentially undetectable bindingagainst these proteins in standard binding assays.

In one embodiment, a high concentration of an anti-CD40 antibody in theaqueous pharmaceutical composition of the invention is at least 50mg/ml. In one embodiment, a high concentration is at least 100 mg/ml. Inone embodiment, a high concentration is at least 150 mg/ml. In oneembodiment, a high concentration is at least 200 mg/ml. In oneembodiment, a high concentration is at least 250 mg/ml. In oneembodiment, a high concentration is at least 300 mg/ml.

In one embodiment, the aqueous pharmaceutical composition of theinvention comprises between 50 mg/ml and 300 mg/ml of an anti-CD40antibody, for example, mAb1.

In one embodiment, the aqueous pharmaceutical composition of theinvention comprises between 75 mg/ml and 250 mg/ml of an anti-CD40antibody, for example, mAb1.

In one embodiment, the aqueous pharmaceutical composition of theinvention comprises between 100 mg/ml and 250 mg/ml of an anti-CD40antibody, for example, mAb1.

In one embodiment, the aqueous pharmaceutical composition of theinvention comprises between 100 mg/ml and 200 mg/ml of an anti-CD40antibody, for example, mAb1.

In one embodiment, the aqueous pharmaceutical composition of theinvention comprises 150 mg/ml of an anti-CD40 antibody, for example,mAb1.

In one embodiment, the aqueous pharmaceutical composition of theinvention comprises about 50 mg/ml, about 60 mg/ml, about 70 mg/ml,about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200mg/ml, about 210 mg/ml, about 220 mg/ml, about 230 mg/ml, about 240mg/ml, about 250 mg/ml or about 300 mg/ml of an anti-CD40 antibody, forexample, mAb1.

Furthermore, the aqueous pharmaceutical compositions are stable suchthat, even after storage for 4 weeks at 2-8° C., less than 5%, 4%, 3%,2%, 1%, 0.05% or 0.01% of the total anti-CD40 antibody is aggregated asmeasured by SEC-HPLC.

The aqueous pharmaceutical compositions may include, in addition to theanti-CD40 antibody, further components such as one or more of thefollowing: (i) a stabiliser; (ii) a buffering agent; (iii) a surfactant;and (iv) a free amino acid. Inclusion of each of such additionalcomponents can give compositions with low aggregation of the anti-CD40antibody.

Suitable stabilisers for use with the invention can act, e.g., asviscosity enhancing agents, bulking agents, solubilising agents, and/orthe like. The stabiliser can be ionic or non-ionic (e.g. sugars). Assugars they include, but are not limited to, monosaccharides, e.g.,fructose, maltose, galactose, glucose, D-mannose, sorbose and the like;disaccharides, e.g. lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, e.g. raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.For example, the sugar may be sucrose, trehalose, raffinose, maltose,sorbitol or mannitol. The sugar may be a sugar alcohol or an aminosugar. Sucrose is particularly useful. As ionic stabiliser they includesalts such as NaCl or amino acid components such as arginine-HCl.

Suitable buffering agents for use with the invention include, but arenot limited to, organic acid salts such as salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid or phtalic acid; Tris, thomethamine hydrochloride, orphosphate buffer. In addition, amino acid components can also be used asbuffering agent. Such amino acid component includes without limitationglycine and histidine. A histidine buffer is particularly useful.

The aqueous pharmaceutical compositions include such buffering agent orpH adjusting agent to provide improved pH control. In one embodiment, anaqueous pharmaceutical composition of the invention has a pH between 5.0and 8.0, between 5.5 and 7.5, between 5.0 and 7.0, between 6.0 and 8.0,or between 6.0 and 7.0. In a specific embodiment, an aqueouspharmaceutical composition of the invention has a pH of about 6.0.

As used herein, the term “surfactant” herein refers to organicsubstances having amphipathic structures; i.e., they are composed ofgroups of opposing solubility tendencies, typically an oil-solublehydrocarbon chain and a water-soluble ionic group. Surfactants can beclassified, depending on the charge of the surface-active moiety, intoanionic, cationic and dispersing agents for various pharmaceuticalcompositions and preparations of biological materials.

Suitable surfactants for use with the invention include, but are notlimited to, non-ionic surfactants, ionic surfactants and zwitterionicsurfactants. Typical surfactants for use with the invention include, butare not limited to, sorbitan fatty acid esters (e.g. sorbitanmonocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitantrioleate, glycerine fatty acid esters (e.g. glycerine monocaprylate,glycerine monomyristate, glycerine monostearate), polyglycerine fattyacid esters (e.g. decaglyceryl monostearate, decaglyceryl distearate,decaglyceryl monolinoleate), polyoxyethylene sorbitan fatty acid esters(e.g. polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monostearate, polyoxyethylenesorbitan monopalmitate, polyoxyethylene sorbitan trioleate,polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol fattyacid esters (e.g. polyoxyethylene sorbitol tetrastearate,polyoxyethylene sorbitol tetraoleate), polyoxyethylene glycerine fattyacid esters (e.g. polyoxyethylene glyceryl monostearate), polyethyleneglycol fatty acid esters (e.g. polyethylene glycol distearate),polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether),polyoxyethylene polyoxypropylene alkyl ethers (e.g. polyoxyethylenepolyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether,polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylenealkylphenyl ethers (e.g. polyoxyethylene nonylphenyl ether),polyoxyethylene hydrogenated castor oils (e.g. polyoxyethylene castoroil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswaxderivatives (e.g. polyoxyethylene sorbitol beeswax), polyoxyethylenelanolin derivatives (e.g. polyoxyethylene lanolin), and polyoxyethylenefatty acid amides (e.g. polyoxyethylene stearic acid amide); C₁₀-C₁₈alkyl sulfates (e.g. sodium cetyl sulfate, sodium lauryl sulfate, sodiumoleyl sulfate), polyoxyethylene C₁₀-C₁₈ alkyl ether sulfate with anaverage of 2 to 4 moles of ethylene oxide units added (e.g. sodiumpolyoxyethylene lauryl sulfate), and C₁-C₁₈ alkyl sulfosuccinate estersalts (e.g. sodium lauryl sulfosuccinate ester); and natural surfactantssuch as lecithin, glycerophospholipid, sphingophospholipids (e.g.sphingomyelin), and sucrose esters of C₁₂-C₁₈ fatty acids. A compositionmay include one or more of these surfactants. Preferred surfactants arepolyoxyethylene sorbitan fatty acid esters e.g. polysorbate 20, 40, 60or 80. Polysorbate 80 (Tween 80) is particularly useful.

Suitable free amino acids for use with the invention include, but arenot limited to, arginine, lysine, histidine, methionine, ornithine,isoleucine, leucine, alanine, glycine, glutamic acid or aspartic acid.The inclusion of a basic amino acid is preferred i.e. arginine, lysineand/or histidine. If a composition includes histidine then this may actboth as a buffering agent and a free amino acid, but when a histidinebuffer is used it is typical to include a non-histidine free amino acide.g. to include histidine buffer and lysine. An amino acid may bepresent in its D- and/or L-form, but the L-form is typical. The aminoacid may be present as any suitable salt e.g. a hydrochloride salt, suchas arginine-HCl.

When present, components (i) to (iv) will be at a concentrationsufficient to maintain the anti-CD40 antibody in a form which is activeand soluble after either

(i) lyophilisation and storage and reconstitution (for lyophilisates),or(ii) conditioning in dosing units and storage (for liquid formulations).

Thus a sugar may be present in the aqueous pharmaceutical composition ofthe invention, e.g. after reconstitution of a lyophilisate in water, ata concentration of between 3 and 400 mM e.g. 50-380 mM, 100-350 mM,200-300 mM. A concentration of 270 mM sucrose is useful.

A buffering agent may be present in the aqueous pharmaceuticalcomposition of the invention, e.g. after reconstitution of alyophilisate in water, at a concentration of between 1 and 60 mM e.g.10-50 mM, 20-40 mM, 25-35 mM. A concentration of 30 mM histidine bufferis useful.

A surfactant may be present in the aqueous pharmaceutical composition ofthe invention, e.g. after reconstitution of a lyophilisate in water, ata concentration of up to 0.2% (by volume) e.g. 0.01-0.1%, 0.03-0.08%,0.04-0.08%. A concentration of 0.06% polysorbate 20 is useful. In someembodiments polysorbate 80 may be used.

A free amino acid may be present in the aqueous pharmaceuticalcomposition of the invention, e.g. after reconstitution of alyophilisate in water, at a concentration of between 2 and 100 mM e.g.10-80 mM, 20-70 mM, 30-60 mM, 40-60 mM. A concentration of 51 mMarginine (e.g. arginine-HCl) or 60 mM methionine or glycine (e.g.glycine-HCl) is useful.

A formulation containing histidine buffer, sucrose and polysorbate 20has been shown to be suitable for lyophilisation of antibody mAb1 at aconcentration of at least 150 mg/ml after reconstitution.

In one embodiment the aqueous pharmaceutical composition consists of 150mg/ml mAb1, 30 mM histidine, 270 mM sucrose and 0.06% polysorbate 20.

In one embodiment the aqueous pharmaceutical composition consists of 150mg/ml mAb1, 30 mM histidine, 270 mM sucrose, 0.06% polysorbate 20 and 51mM arginine-HCl.

In one embodiment the aqueous pharmaceutical composition consists of 150mg/ml mAb1, 30 mM histidine, 270 mM sucrose, 0.06% polysorbate 20 and 60mM glycine-HCl.

In one embodiment the aqueous pharmaceutical composition consists of 200mg/ml mAb1, 30 mM histidine, 270 mM sucrose, and 0.06% polysorbate 20.

In one embodiment the aqueous pharmaceutical composition consists of 200mg/ml mAb1, 30 mM histidine, 270 mM sucrose, 0.06% polysorbate 20 and 51mM arginine-HCl.

In one embodiment the aqueous pharmaceutical composition consists of 75mg/ml mAb1, 30 mM histidine, 270 mM sucrose, and 0.06% polysorbate 20.

In one embodiment the aqueous pharmaceutical composition consists of 75mg/ml mAb1, 30 mM histidine, 270 mM sucrose, 0.06% polysorbate 20 and 51mM arginine-HCl.

Other contemplated excipients, which may be utilised in the aqueouspharmaceutical compositions of the invention include, for example,flavoring agents, antimicrobial agents, sweeteners, antioxidants,antistatic agents, lipids such as phospholipids or fatty acids, steroidssuch as cholesterol, protein excipients such as serum albumin (humanserum albumin), recombinant human albumin, gelatin, casein, salt-formingcounterions such sodium and the like. These and additional knownpharmaceutical excipients and/or additives suitable for use in theformulations of the invention are known in the art, e.g., as listed in“The Handbook of Pharmaceutical Excipients, 4^(th) edition, Rowe et al.,Eds., American Pharmaceuticals Association (2003); and Remington: theScience and Practice of Pharmacy, 21^(th) edition, Gennaro, Ed.,Lippincott Williams & Wilkins (2005).

The aqueous pharmaceutical compositions of the invention may includefurther active ingredients in addition to the anti-CD40 antibody.Further pharmacological agents may include, for instance,chemotherapeutic compounds.

Lyophilisates

Techniques for lyophilisation of antibodies are well known in the arte.g. see John F. Carpenter and Michael J. Pikal, 1997 (Pharm. Res. 14,969-975); Xialin (Charlie) Tang and Michael J. Pikal, 2004 (Pharm. Res.21, 191-200). For example, the monoclonal antibody products SYNAGIS™,REMICADE™, RAPTIVA™, SIMULECT™, XOLAIR™ and HERCEPTIN™ are supplied aslyophilisates. These antibodies are reconstituted to various finalconcentrations e.g. SIMULECT™ is reconstituted to a concentration of 4mg/ml antibody, REMICADE™ is reconstituted to a concentration of 10mg/ml, HERCEPTIN™ to 21 mg/ml, SYNAGIS™ and RAPTIVA™ to 100 mg/ml, andXOLAIR™ to 125 mg/ml.

Pre-Lyophilisates, Lyophilisates and Aqueous Reconstitution

Before a lyophilisate can be administered to a patient it should bereconstituted with an aqueous reconstituent. This step permits antibodyand other components in the lyophilisate to re-dissolve to give asolution which is suitable for injection to a patient.

The volume of aqueous material used for reconstitution dictates theconcentration of the antibody in a resulting pharmaceutical composition.Reconstitution with a smaller volume of reconstituent than thepre-lyophilisation volume provides a composition which is moreconcentrated than before lyophilisation. The reconstitution factor(volume of formulation after lyophilisation:volume of formulationbefore) may be from 1:0.5 to 1:6. A reconstitution factor of 1:3 isuseful. As mentioned above, lyophilisates of the invention can bereconstituted to give aqueous compositions with an anti-CD40 antibodyconcentration of at least 50 mg/ml, 100 mg/ml, 150 mg/ml. 200 mg/ml, 250mg/ml or 300 mg/ml, and the volume of reconstituent will be selectedaccordingly. If required, the reconstituted formulation can be dilutedprior to administration to a patient as appropriate to deliver theintended dose.

Typical reconstituents for lyophilised antibodies include sterile wateror buffer, optionally containing a preservative. If the lyophilisateincludes a buffering agent then the reconstituent may include furtherbuffering agent (which may be the same as or different from thelyophilisate's buffering agent) or it may instead include no bufferingagent (e.g. WFI (water for injection), or physiological saline).

When present, components (i) to (iv) will be at a pre-lyophilisationconcentration sufficient to maintain the anti-CD40 antibody in a formwhich is active and soluble after storage (under normal conditions) andreconstitution. The components will also be present afterreconstitution.

Thus a sugar, such as sucrose or trehalose, may be present beforelyophilisation at a concentration of between 3 and 300 mM e.g. 15-200mM, 30-150 mM, 80-100 mM. A concentration of 90 mM sucrose is useful. Abuffering agent, such as histidine, may be present before lyophilisationat a concentration of between 1 and 60 mM e.g. 3-30 mM, 5-20 mM, 5-15mM. A concentration of 10 mM histidine buffer is useful. A surfactant,such as polysorbate 80 or polysorbate 20 may be present beforelyophilisation at a concentration of up to 0.2% (by volume) e.g.0.01-0.1%, 0.01-0.08%, 0.01-0.04%. A concentration of 0.02% polysorbate80 or polysorbate 20 is useful. A free amino acid, such as arginine,methionine or glycine, may be present before lyophilisation at aconcentration of between 2 and 80 mM e.g. 3-60 mM, 3-50 mM, 6-30 mM,10-25 mM, 15-20 mM. A concentration of 17 mM arginine-HCl or 20 mMglycine-HCl or 60 mM methionine is useful. The anti-CD40 antibody ispresent before lyophilisation at a concentration of between 20 mg/ml and120 mg/ml, e.g. 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 66.6mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, or 120 mg/ml.A concentration of 50 mg/ml is useful.

The pre-lyophilisate of the invention has a pH between 5.0 and 8.0,between 5.0 and 7.0, between 5.5 and 6.5. In a specific embodiment, thepre-lyophilisate of the invention has a pH of about 6.0.

In one embodiment the pre-lyophilisate of the invention has a molarratio of sucrose:antibody of 90:1 and a molar ratio ofhistidine:antibody of 10:1.

In one embodiment the pre-lyophilisate of the invention has a molarratio of sucrose:antibody of 90:1, a molar ratio of histidine:antibodyof 10:1, and a molar ratio of arginine-HCl:antibody of 17:1.

In one embodiment the pre-lyophilisate of the invention has a molarratio of sucrose:antibody of 90:1, a molar ratio of histidine:antibodyof 10:1, and a molar ratio of glycine-HCl:antibody of 60:1.

A formulation containing histidine buffer, sucrose, polysorbate 20 and,optionally arginine, methionine or glycine has been shown to be suitablefor lyophilisation of antibody mAb1. After reconstitution, thecomponents of the lyophilisate may be present at a concentration of theaqueous pharmaceutical compositions as described hereinbefore.

Target Diseases and Disorders

The aqueous pharmaceutical compositions of the invention comprisinganti-CD40 antibodies can be used to treat, ameliorate or preventCD40-related autoimmune disorders, CD40-related inflammatory disordersand/or to prevent or reduce the risk of graft rejection intransplantation. For the purposes of the present invention, the term“inflammatory disorders” includes “autoimmune disorders”.

As used herein, the term “autoimmunity” is generally understood toencompass inflammatory immune-mediated processes involving “self”antigens. In autoimmune diseases, self antigen(s) trigger host immuneresponses.

Pharmaceutical compositions comprising anti-CD40 antibodies areparticularly useful to treat Multiple Sclerosis, Systemic LupusErythematosus, Sjögren's syndrome, Rheumatoid Arthritis, transplantrejection; graft-versus-host disease, Pemphigus vulgaris; and B cellneoplasms such as acute lymphoblastic leukemia (ALL) and B-cell chroniclymphocytic leukemia (CLL).

Also, the present invention includes treatment of inflammationassociated with tissue transplant rejection. “Transplant rejection” or“graft rejection” refers to any host-mounted immune response against agraft including but not limited to HLA antigens, blood group antigens,and the like.

The invention can also be used to treat graft versus host disease, suchas that associated with bone marrow transplantation, for example. Insuch graft versus host disease, the donor bone marrow includeslymphocytes and cells that mature into lymphocytes. The donor'slymphocytes recognise the recipient's antigens as non-self and mount aninflammatory immune response. Hence, as used herein, “graft versus hostdisease” or “graft versus host reaction” refers to any T cell mediatedimmune response in which donor lymphocytes react to the host's antigens.

The antagonist anti-CD40 antibodies or proteins described herein, forexample mAb1, mAb2 or mAb3, can be used in accordance with the methodsof the invention to treat autoimmune and/or inflammatory disordersincluding, but not limited to, systemic lupus erythematosus (SLE),discoid lupus, lupus nephritis, sarcoidosis, inflammatory arthritis,including juvenile arthritis, rheumatoid arthritis, psoriatic arthritis,Reiter's syndrome, ankylosing spondylitis, and gouty arthritis,rejection of an organ or tissue transplant, hyperacute, acute, orchronic rejection and/or graft versus host disease, multiple sclerosis,hyper IgE syndrome, polyarteritis nodosa, primary biliary cirrhosis,inflammatory bowel disease, Crohn's disease, celiac's disease(gluten-sensitive enteropathy), primary Sjögren's syndrome (pSS),autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia,psoriasis, scleroderma, myasthenia gravis, autoimmune thrombocytopenicpurpura, autoimmune thyroiditis, Grave's disease, Hasimoto'sthyroiditis, immune complex disease, chronic fatigue, immune dysfunctionsyndrome (CFIDS), polymyositis and dermatomyositis, cryoglobulinemia,thrombolysis, cardiomyopathy, pemphigus vulgaris, pulmonary interstitialfibrosis, Type I and Type II diabetes mellitus, type 1, 2, 3, and 4delayed-type hypersensitivity, allergy or allergic disorders,unwanted/unintended immune responses to therapeutic proteins (see forexample, U. S. Patent Application No. US 2002/0119151 and Koren, et al.(2002) Curr. Pharm. Biotechnol. 3: 349-60), asthma, Churg-Strausssyndrome (allergic granulomatosis), atopic dermatitis, allergic andirritant contact dermatitis, urtecaria, IgE-mediated allergy,atherosclerosis, ANCA-associated Vasculitides, vasculitis, idiopathicinflammatory myopathies, hemolytic disease, Alzheimer's disease, chronicinflammatory demyelinating polyneuropathy, and the like.

Genetic ablation or pharmacological inhibition of the CD40-CD154 pathwayhas previously demonstrated therapeutic benefit in either the clinic orin preclinical models of SLE, pSS, ITP, MS, Crohn's disease, Pemphigusvulgaris, autoimmune vasculitis and RA (Law C L, Grewal I S. (2009).Adv. Exp. Med. Biol. 2009; 647:8-36); the medical need of which isdetailed below.

In preferred embodiments the anti-CD40 antibodies or proteins of theinvention are useful in treating: (i) systemic lupus erythematosus(lupus nephritis), preferably in providing effective steroid-sparingtherapies for induction and maintenance of remission, and prevention ofend-stage renal disease; (ii) primary Sjögren's syndrome, preferably inprevention of salivary and lacrimary gland destruction, and inductionand maintenance of remission of extraglandular manifestations; (iii)autoimmune thrombocytopenic purpura, preferably treatment of patientsrefractory to standard of care; (iv) ANCA-associated Vasculitides,preferably inducing and maintaining remission in patients refractory tocorticosteroids, and steroid-sparing treatment; (v) Pemphigus Vulgaris,preferably in induction and maintenance of remission in patientsrefractory to corticosteroids, and steroid-sparing treatment; (vi)Multiple Sclerosis, preferably in providing more effective treatmentsfor prevention of relapses and disability progression, and achievingdisease-free status; and (vii) Crohn's disease, preferably in providingmore effective therapies for maintenance of remission, and treatment ofpatients refractory to anti-TNF.

In some other embodiments, the anti-CD40 antibodies or proteins of theinvention are useful in treating pulmonary inflammation including butnot limited to lung graft rejection, asthma, sarcoidosis, emphysema,cystic fibrosis, idiopathic pulmonary fibrosis, chronic bronchitis,allergic rhinitis and allergic diseases of the lung such ashypersensitivity pneumonitis, eosinophilic pneumonia, bronchiolitisobliterans due to bone marrow and/or lung transplantation or othercauses, graft atherosclerosis/graft phlebosclerosis, as well aspulmonary fibrosis resulting from collagen, vascular, and autoimmunediseases such as rheumatoid arthritis, scleroderma and lupuserythematosus.

“Treatment” is herein defined as the application or administration of ananti-CD40 antibody or protein according to the invention, for example,mAb1, mAb2 or mAb3 antibody, to a subject, or application oradministration a pharmaceutical composition comprising said anti-CD40antibody or protein of the invention to an isolated tissue or cell linefrom a subject, where the subject has an autoimmune disease and/orinflammatory disease, a symptom associated with an autoimmune diseaseand/or inflammatory disease, or a predisposition toward development ofan autoimmune disease and/or inflammatory disease, where the purpose isto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,or affect the autoimmune disease and/or inflammatory disease, anyassociated symptoms of the autoimmune disease and/or inflammatorydisease, or the predisposition toward the development of the autoimmunedisease and/or inflammatory disease.

By “treatment” is also intended the application or administration of apharmaceutical composition comprising an anti-CD40 antibodies or proteinof the invention, for example, mAb1, mAb2 or mAb3 antibody, to asubject, or application or administration of a pharmaceuticalcomposition comprising said anti-CD40 antibody or protein of theinvention to an isolated tissue or cell line from a subject, where thesubject has an autoimmune disease and/or inflammatory disease, a symptomassociated with an autoimmune disease and/or inflammatory disease, or apredisposition toward development of an autoimmune disease and/orinflammatory disease, where the purpose is to cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve, or affect the autoimmunedisease and/or inflammatory disease, any associated symptoms of theautoimmune disease and/or inflammatory disease, or the predispositiontoward the development of the autoimmune disease and/or inflammatorydisease.

By “anti-inflammatory activity” is intended a reduction or prevention ofinflammation. Therapy with at least one anti-CD40 antibody or proteinaccording to the invention causes a physiological response that isbeneficial with respect to treatment of an autoimmune disease and/orinflammatory disease, where the disease involves cells expressing theCD40 antigen. It is recognised that the methods of the invention may beuseful in preventing phenotypic change in cells such as proliferation,activation, and the like.

Patient Administration

A pharmaceutical composition of the invention can be administered to apatient. Administration will typically be via a syringe. Thus theinvention provides a delivery device (e.g. a syringe) including apharmaceutical composition of the invention (e.g., pre-filled syringe).Patients will receive an effective amount of the anti-CD40 antibody asthe principal active ingredient i.e. an amount that is sufficient totreat, ameliorate, or prevent the disease or disorder in question.Therapeutic effects may also include reduction in physical symptoms. Theoptimum effective amount and concentration of antibody for anyparticular subject will depend upon various factors, including thepatient's age size health and/or gender, the nature and extent of thecondition, the activity of the particular antibody, the rate of itsclearance by the body, and also on any possible further therapeutic(s)administered in combination with the antibody. The effective amountdelivered for a given situation can be determined within the judgment ofa clinician. For purposes of the present invention, an effective dosemay be from about 0.005 mg/kg to about 50 mg/kg, or about 0.05 mg/kg toabout 10 mg/kg. Known antibody-based pharmaceuticals provide guidance inthis respect e.g. HERCEPTIN™ is administered with an initial loadingdose of 4 mg/kg body weight and a weekly maintenance dose of 2 mg/kgbody weight; RITUXAN™ is administered weekly at 375 mg/m²; SYNAGIS™ isadministered intramuscularly at 15 mg/kg body weight; etc.

The invention provides a method for delivering a monoclonal antibody toa mammal, comprising a step of administering to the patient apharmaceutical composition of the invention.

The invention also provides a method for delivering a monoclonalantibody to a mammal, comprising steps of: (i) reconstituting alyophilisate of the invention to give an aqueous formulation, and (ii)administering the aqueous formulation to the patient. Step (ii) ideallytakes place within 24 hours of step (i) e.g. within 12 hours, within 6hours, within 3 hours, or within 1 hour.

The invention also provides formulations of the invention for use asmedicaments e.g. for use in delivering an antibody to a mammal, or foruse in treating, preventing or ameliorating one or more of the diseasesand disorders described above.

The mammal is preferably a human but may also be, for example, a horseor a cow or a dog or a cat. The antibodies will ideally be chosen tomatch the target species e.g. a human antibody for human administration,an equine antibody for horses, a canine antibody for dogs, etc. Ifnative host antibodies are not available then transfer of antibodyspecificity from one species to another can be achieved by transfer ofCDR residues (and typically, in addition, one or more frameworkresidues) from a donor antibody into a recipient framework from the hostspecies e.g. as in humanisation. Equinised, bovinised, caninised andfelinised antibodies are known in the art. The antibody will bind toCD40 from the target species, but it may also cross-react with CD40 fromother species.

Dosage can be by a single dose schedule or a multiple dose schedule.

Ingredients for forming compositions of the invention (e.g.lyophilisates and reconstituents) may be supplied in hermetically-sealedcontainers.

The Anti-CD40 Antibody

The invention concerns the formulation of anti-CD40 antibodies and morespecifically the antibodies designated mAb1, mAb2, and mAb3, especiallymAb1.

One suitable antibody that can be comprised in the pharmaceuticalcompositions of the invention is the human recombinant antibody mAb1,structurally characterised as further described below. The V_(H) aminoacid sequence of such isolated anti-CD40 antibody is shown in SEQ IDNO: 1. The V_(L) amino acid sequence of such isolated anti-CD40 antibodyis shown in SEQ ID NO: 2. The full length heavy chain amino acidsequence of such isolated anti-CD40 antibody is shown in SEQ ID NO: 9.The full-length light chain amino acid sequence of such isolatedanti-CD40 antibody is shown in SEQ ID NO: 10. Another example of heavyand light chain amino acid sequences of such isolated anti-CD40antibodies are those encoded by the nucleotide sequences of SEQ ID NO:11 and SEQ ID NO: 12 respectively.

Other anti-CD40 antibodies that can be used for preparing thepharmaceutical compositions of the invention include anti-CD40antibodies with amino acids that have been mutated by amino aciddeletion, insertion or substitution, yet have no more than 1, 2, 3, 4 or5 amino acid deletions, insertions or substitutions in either the heavyor light chain regions described above. In a specific embodiment, suchamino acid changes appear only within the framework and/or constantregions and the CDR regions are 100% identical to the heavy chain CDR1,CDR2 and CDR3 regions of SEQ ID NO: 3, 4 and 5 and to the light chainCDR1, CDR2 and CDR3 regions of SEQ ID NO: 6, 7, and 8 respectively. Inone more specific embodiment, the changes that have been made are onlyconservative amino acid substitutions outside of the CDR regions.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues outside of the CDR regions of an anti-CD40antibody, can be replaced with other amino acid residues from the sameside chain family, and the altered antibody can be tested for retainedfunction, in particular the same binding properties to CD40.

Antibodies may typically be glycosylated. N-linked glycans attached tothe C_(H)2 domain of a heavy chain, for instance, can influence C1q andFcR binding, and aglycosylated antibodies (for example comprising anN297A mutation) may have lower or different affinity for thesereceptors. The glycan structure can also affect activity e.g.differences in complement-mediated cell death may be seen depending onthe number of galactose sugars (0, 1 or 2) at the terminus of a glycan'sbiantennary chain. An antibody's glycans preferably do not lead to ahuman immunogenic response after administration.

Another modification of the anti-CD40 antibodies herein that iscontemplated by the invention is pegylation. An antibody can bepegylated to, for example, increase the biological (e.g., serum)half-life of the antibody. To pegylate an antibody, the antibody, orfragment thereof, typically may be reacted with polyethylene glycol(PEG), such as a reactive ester or aldehyde derivative of PEG, underconditions in which one or more PEG groups become attached to theantibody or antibody fragment. The pegylation can be carried out by anacylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer).

As used herein, the term “polyethylene glycol” is intended to encompassany of the forms of PEG that have been used to derivatise otherproteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycolor polyethylene glycol-maleimide. In certain embodiments, the antibodyto be pegylated is an aglycosylated antibody. Methods for pegylatingproteins are known in the art and can be applied to the antibodies ofthe invention. See for example, EP 0 154 316 by Nishimura et al. and EP0 401 384 by Ishikawa et al.

Any other natural or non-natural post-translational modification ofanti-CD40 antibodies (e.g. mAb1) is further contemplated as specificembodiments of anti-CD40 antibodies that could be used for preparing thepharmaceutical compositions of the invention.

Antibodies can be prepared in a form free from products with which theywould naturally be associated. Contaminant components of an antibody'snatural environment include materials such as enzymes, hormones, orother host cell proteins.

The various features and embodiments of the present invention, referredto in individual sections and embodiments above apply, as appropriate,to other sections and embodiments, mutatis mutandis. Consequentlyfeatures specified in one section or embodiment may be combined withfeatures specified in other sections or embodiments, as appropriate.

EXAMPLES Preparing Anti-CD40 Antibodies

CHIR-12.12, and mAb1 (N297A CHIR-12.12), mAb2 (D265A CHIR-12.12), andmAb3 (CHIR-12.12 LALA) bind specifically to CD40. Tables 1 and 2 belowsummarise the sequence characteristics of these antibodies. Theseantibodies may be produced in mammalian host cells, such as, a CHO cellline transfected with expression vectors carrying heavy and light chaincoding sequences under suitable expression promoters.

TABLE 1 Brief description of the sequences listed in the sequencelisting of Table 2 SEQ ID NO: Description of the sequence 1 Amino acidsequence of the variable region (V_(H)) of the heavy chain of CHIR-12.12, mAb1, mAb2 and mAb3 2 Amino acid sequence of the variable region(V_(L)) of the light chain of CHIR- 12.12, mAb1, mAb2 and mAb3 3 Aminoacid sequence of HCDR1 of CHIR-12.12, mAb1, mAb2 and mAb3 4 Amino acidsequence of HCDR2 of CHIR-12.12, mAb1, mAb2 and mAb3 5 Amino acidsequence of HCDR3 of CHIR-12.12, mAb1, mAb2 and mAb3 6 Amino acidsequence of LCDR1 of CHIR-12.12, mAb1, mAb2 and mAb3 7 Amino acidsequence of LCDR2 of CHIR-12.12, mAb1, mAb2 and mAb3 8 Amino acidsequence of LCDR3 of CHIR-12.12, mAb1, mAb2 and mAb3 9 Amino acidsequence of the full length heavy chain of mAb1 10 Amino acid sequenceof the full length light chain of mAb1 11 Nucleotide sequence encodingfull length heavy chain of mAb1 (SEQ ID NO: 9) 12 Nucleotide sequenceencoding full length light chain of mAb1 (SEQ ID NO: 10) 13 Amino acidsequence of the full length heavy chain of mAb2 14 Amino acid sequenceof the full length light chain of mAb2 15 Nucleotide sequence encodingfull length heavy chain of mAb2 (SEQ ID NO: 13) 16 Nucleotide sequenceencoding full length light chain of mAb2 (SEQ ID NO: 14) 17 Amino acidsequence of the full length heavy chain of mAb3 18 Amino acid sequenceof the full length light chain of mAb3 19 Nucleotide sequence encodingfull length heavy chain of mAb3 (SEQ ID NO: 17) 20 Nucleotide sequenceencoding full length light chain of mAb3 (SEQ ID NO: 18) 21 Amino acidsequence of human CD40

TABLE 2  Sequence listing SEQ ID NO: Amino acid or Nucleotide Sequence 1QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIA APGPDYWGQGTLVTVSS2 DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFG PGTKVDIR 3 SYGMH 4VISYEESNRYHADSVKG 5 DGGIAAPGPDY 6 RSSQSLLYSNGYNYLD 7 LGSNRAS 8 MQARQTPFT9 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 10DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 11CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGGTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGGCATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAGATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACGCCTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCACAGGTGTACACACTGCCCCCCAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTCCAACGGACAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 12GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCGAGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCACAGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCGGTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGACCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGA GCTTCAACAGGGGCGAGTGC13 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PV TKSFNRGEC 15CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGGTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGGCATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAGATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCACAGGTGTACACACTGCCCCCCAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTCCAACGGACAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 16GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCGAGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCACAGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCGGTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGACCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGA GCTTCAACAGGGGCGAGTGC17 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 18DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 19CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGGTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGGCATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAGATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCC CTGTCCCTGTCTCCCGGCAAG20 GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCGAGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCACAGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCGGTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGACCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGA GCTTCAACAGGGGCGAGTGC21 MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVT QEDGKESRISVQERQ

Examples of Formulations

A high concentration lyophilised or liquid formulation of mAb1 wasdesired and so formulation studies were performed. A lyophilisedformulation comprising a sugar, a buffering agent and a surfactant wasstable and could maintain high antibody concentrations afterreconstitution.

Five formulations (F1, F2, F3, F4 and F5) of mAb1 were evaluated forstability. F1 was a liquid formulation at 50 mg/mL mAb1 at pH 6.0.Formulations F2, F3, F4, and F5 had prior to lyophilisation, 50 mg/vialmAb1. Formulations F2, F3, F4 and F5 had, prior to lyophilisation, 50mg/ml mAb1 at pH 6.0. Formulations F2, F3, F4 and F5 had a fill volumeof 3.6 ml. The five formulations included buffer, sugar, surfactant andfree amino acid as listed in Table 3:

TABLE 3 Examples of formulations Histidine Amino mAb1 buffer StabiliserSurfactant acid F1 50 mg/ml 10 mM 190 mM 0.02% polysorbate 40 mMtrehalose 20 arginine- HCl F2 50 mg/ml 10 mM 90 mM 0.02% polysorbate 17mM trehalose 20 arginine- HCl F3 50 mg/ml 10 mM 90 mM 0.02% polysorbate17 mM sucrose 20 arginine- HCl F4 50 mg/ml 10 mM 90 mM 0.02% polysorbate— trehalose 20 F5 50 mg/ml 10 mM 90 mM 0.02% polysorbate — sucrose 20

The F2, F3, F4, and F5 lyophilisates were reconstituted with WFI (1.0ml) to give a reconstituted volume of 1.2 ml (⅓ the original aqueousvolume). The reconstituted compositions were as listed in Table 4:

TABLE 4 Examples of formulations Histidine Amino mAb1 buffer StabiliserSurfactant acid F2 150 mg/ml 30 mM 270 mM 0.06% polysorbate 51 mMhistidine trehalose 20 arginine- HCl F3 150 mg/ml 30 mM 270 mM 0.06%polysorbate 51 mM histidine sucrose 20 arginine- HCl F4 150 mg/ml 30 mM270 mM 0.06% polysorbate — histidine trehalose 20 F5 150 mg/ml 30 mM 270mM 0.06% polysorbate — histidine sucrose 20

The lyophilisation cycle used is reported in Table 5.

TABLE 5 The lyophilisation cycle parameters Time Chamber Step Operation[hh:mm] Shelf temp. pressure 1 Vial loading As 20° C. Ambient required 25° C. 00:30  5° C. Ambient cooling 3 5° C. hold 03:00  5° C. Ambient 4Freeze ramp 01:24   5° C. to −37° C. Ambient 5 Freeze hold 06:00 −37° C.Ambient 6 Chamber as −37° C. 0.2 mbar^(a) Vacuum required 7 Primary16:00 −37° C. to 25° C. 0.2 mbar^(a) drying ramp 8 Secondary 24:00 25°C. 0.2 mbar^(a) drying hold 11 Vial 25° C. 850 ± 50 mbar stoppering^(a)Chamber pressure was controlled using sterile filtered nitrogn. Thepressure was determined by instruments based on capacitancemeasurements.

The five formulations were tested for stability at various time pointsafter formulation/reconstitution as listed below.

Size exclusion-High Pressure Liquid Chromatography (SEC-HPLC) was usedto assess the amount of mAb1 in each formulation at the time offormulation (T0), 4 weeks (25° C.), 4 weeks (40° C.), 6 months (2-8°C.), 6 months (25° C.) and 6 months (40° C.). The amount of antibody isexpressed as a percentage of the starting amount, as listed in Table 6.The F1 formulation remains close to 50 mg/ml±5 mg/ml (100%±10%), the F2,F3, F4 and F5 formulations remain close to 150 mg/ml±15 mg/ml(100%±10%).

TABLE 6 Amount of antibody from SEC-HPLC 4 weeks 4 weeks 6 months 6months 6 months T0 (25° C.) (40° C.) (2-8° C.) (25° C.) (40° C.) F1 100%± 10% 100% ± 10% 100% ± 10% 100% ± 10% 100% ± 10% 100% ± 10% F2 100% ±10% — 100% ± 10% — 100% ± 10% 100% ± 10% F3 100% ± 10% — 100% ± 10% — —100% ± 10% F4 100% ± 10% — 100% ± 10% — 100% ± 10% 100% ± 10% F5 100% ±10% — 100% ± 10% — 100% ± 10% 100% ± 10%

The potency of the mAb1 antibody in each formulation was measured byELISA assay at T0, 4 weeks (25° C.), 4 weeks (40° C.), 6 months (2-8°C.), 6 months (25° C.) and 6 months (40° C.). The results, expressed asa percentage of the starting potency (100%) are listed in Table 7. Thepotency of the F1 formulation decreases with progression of time andincrease of temperature; after 6 m at 40° C. potency was found to be79%. The lowest value measured was 85% for F4 (6 m at 25° C.) and 86%for F3 (6 m at 40° C.)

TABLE 7 Potency of antibody as measured in ELISA 4 weeks 4 weeks 6months 6 months 6 months T0 (25° C.) (40° C.) (2-8° C.) (25° C.) (40°C.) F1 91.3 104.0 98.0 91.0 91.0 79.0 F2 93.2 F3 95.7 86.0 F4 96.5 85.090.0 F5 97.8

Stability of the formulations was evaluated by % impurities as measuredby size exclusion-High Pressure Liquid Chromatography (SEC-HPLC) andDynamic Light Scattering (DLS). The results are shown in Tables 8 and 9.

TABLE 8 Aggregation products results from SEC-HPLC 4 weeks 4 weeks 6months 6 months 6 months T0 (25° C.) (40° C.) (2-8° C.) (25° C.) (40°C.) F1 0.26% 0.26% 0.59% 0.38% 0.47% 2.76% F2 0.38% 1.37% 1.40% 3.80% F30.29% 0.96% 2.88% F4 0.38% 1.94% 1.97% 5.59% F5 0.33% 1.32% 1.40% 4.01%

TABLE 9 Small particles; results for PolyDispensity Index (PDI) fromDynamic Light Scattering 4 weeks 4 weeks 6 months 6 months 6 months T0(25° C.) (40° C.) (2-8° C.) (25° C.) (40° C.) F1 52.8% 30.2% 39.3% 41.3%52.9% 59.9% F2 48.8% 61.0% 44.4% 46.6% F3 50.4% 57.0% 40.8% F4 54.7%61.8% 61.9% 59.2% F5 47.2% 60.0% 60.5% 51.3%

The F1 formulation showed an increase of aggregation products withincreasing temperature and time. For the F2 to F5 formulations additionof arginine (F2 and F3) slightly reduced the level of aggregation; andsucrose (F3 and F5) was superior to trehalose.

Aggregation products were also evaluated by measuring turbidity. The F1formulation showed an increase of turbidity with increase in temperatureand with increase in time. The F2 to F5 formulations showed slightlyincreased turbidity values for formulations containing arginine (F2 andF3).

TABLE 10 Turbidity measurements (absorbance units) 4 weeks 4 weeks 6months 6 months 6 months T0 (25° C.) (40° C.) (2-8° C.) (25° C.) (40°C.) F1 7.38 11.30 12.90 9.7 12.7 17.9 F2 9.36 14.90 12.1 11.3 F3 8.9814.40 11.0 F4 6.34 10.40 9.2 7.6 F5 6.91 10.70 9.7 8.2

Stability of the formulations was evaluated by % degradation products asmeasured SEC-HPLC. With the F2 to F5 formulations the addition ofarginine slightly reduced degradation. The results are shown in Table11.

TABLE 11 Degradation products results from SEC-HPLC 4 weeks 4 weeks 6months 6 months 6 months T0 (25° C.) (40° C.) (2-8° C.) (25° C.) (40°C.) F1 0.11% 1.40% 2.76% 1.04% 2.10% 8.88% F2 0.11% 0.14% 0.12% 0.05% F30.15% 0.18% 0.00% 2.97% F4 0.10% 0.19% 0.15% 5.70% F5 0.11% 0.18% 0.14%4.10%

The four lyophilised formulations F2, F3, F4, and F5 were assessed forvisual clarity after reconstitution. The results are presented in Table12.

TABLE 12 Visual clarity 4 weeks 4 weeks 6 months 6 months 6 months T0(25° C.) (40° C.) (2-8° C.) (25° C.) (40° C.) F2 clear clear clear clearclear clear without without without without without without particlesparticles particles particles particles particles F3 clear clear clearclear clear clear without without without without without withoutparticles particles particles particles particles particles F4 clearclear clear clear clear clear without without without without withoutwithout particles particles particles particles particles particles F5clear clear clear clear clear clear without without without withoutwithout without particles particles particles particles particlesparticles

The physico-chemical properties of the formulations were assessed; theresults are presented in Table 13. The viscosity values are the valuesof the formulations having 50 mg/ml antibody prior to lyophilisation.The osmolality values correspond to the reconstituted compositionshaving 150 mg/ml antibody.

TABLE 13 Physico-chemical properties Viscosity Osmolality mPa · smOsm/kg F1 2.1 318 F2 1.7 580 F3 1.6 556 F4 1.6 461 F5 1.6 443

CONCLUSIONS

Overall, the formulations tested showed good results. The bioassays (SECand ELISA) showed that the formulations are comparable. Solubleaggregates were slightly reduced in the presence of arginine and wereslightly lower for the sucrose containing formulations compared to thetrehalose containing formulations. Turbidity was slightly increased inthe presence of arginine; there was no difference between trehalose orsucrose containing formulations. Degradation was reduced in the presenceof arginine. With regard to physico-chemical properties the pH,viscosity, residual moisture content, and visual aspects were comparablefor all formulations. Osmolality was >300 mOsm/kg for all formulations,and was even higher in the presence of arginine-HCl (F2 and F3). It isdesirable to have a formulation which is isotonic to plasma (290mOsm/kg), or as close to this value as possible. The formulations havingsucrose (F3 and F5) were considered preferable to those comprisingtrehalose. Whilst some beneficial effects on degradation were seen witharginine (F3), the lower osmolality of the F5 formulation was consideredto be a more important factor. The F5 formulation was deemed to be themost optimal formulation.

1. A lyophilised formulation prepared by lyophilising an aqueousformulation having a pH of 5.0-7.0 and comprising (i) an anti-CD40antibody wherein the antibody has a concentration of 20-150 mg/ml, andwherein said anti-CD40 antibody includes heavy chain CDR1, CDR2 and CDR3of SEQ ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2 andCDR3 of SEQ ID NOs: 6, 7 and 8, (ii) a stabiliser, (iii) a bufferingagent, (iv) a surfactant, and optionally (v) an amino acid.
 2. Thelyophilised formulation of claim 1, wherein said formulation is preparedfrom an aqueous formulation having a pH of 5.0-7.0 and comprising (i) ananti-CD40 antibody wherein the antibody has a concentration of 20-150mg/ml, and wherein said anti-CD40 antibody includes heavy chain CDR1,CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and light chainCDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8, (ii) sucrose or trehaloseas a stabiliser, (iii) histidine as a buffering agent, (iv) polysorbate20 as a surfactant, and optionally (v) an amino acid selected fromarginine, methionine and glycine.
 3. The lyophilised formulation ofclaim 2, wherein said formulation is prepared from an aqueousformulation having a pH of 5.0-7.0 and comprising (i) an anti-CD40antibody wherein the antibody has a concentration of 20-150 mg/ml, andwherein said anti-CD40 antibody includes heavy chain CDR1, CDR2 and CDR3of SEQ ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2 andCDR3 of SEQ ID NOs: 6, 7 and 8, (ii) 3-300 mM sucrose or trehalose as astabiliser, (iii) 1-60 mM histidine as a buffering agent, (iv) up to0.2% polysorbate 20 as a surfactant, and optionally (v) 2-80 mMarginine, methionine or glycine.
 4. The lyophilised formulation asclaimed in claim 3, wherein said formulation is prepared from an aqueousformulation having a pH of 6.0 and comprising (i) an anti-CD40 antibodywherein the antibody has a concentration of 50 mg/ml, and wherein saidanti-CD40 antibody includes heavy chain CDR1, CDR2 and CDR3 of SEQ IDNOs 3, 4 and 5 respectively, and light chain CDR1, CDR2 and CDR3 of SEQID NOs: 6, 7 and 8, (ii) 90 mM sucrose as a stabiliser, (iii) 10 mMhistidine as a buffering agent, (iv) 0.02% polysorbate 20 as asurfactant.
 5. The lyophilised formulation as claimed in claim 3,wherein said formulation is prepared from an aqueous formulation havinga pH of 6.0 and comprising (i) an anti-CD40 antibody wherein theantibody has a concentration of 50 mg/ml, and wherein said anti-CD40antibody includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and5 respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7and 8, (ii) 90 mM sucrose as a stabiliser, (iii) 10 mM histidine as abuffering agent, (iv) 0.02% polysorbate 20 as a surfactant, and (v) 17mM arginine.
 6. The lyophilised formulation as claimed in claim 3,wherein said formulation is prepared from an aqueous formulation havinga pH of 6.0 and comprising (i) an anti-CD40 antibody wherein theantibody has a concentration of 150 mg/ml, and wherein said anti-CD40antibody includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and5 respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7and 8, (ii) 270 mM sucrose as a stabiliser, (iii) 30 mM histidine as abuffering agent, and (iv) 0.06% polysorbate 20 as a surfactant.
 7. Anaqueous pharmaceutical composition obtained by reconstituting alyophilised formulation of claim 1, wherein the reconstitution factor isbetween 1:0.5 to 1:6.
 8. The aqueous pharmaceutical composition of claim8, wherein the reconstitution factor is 1:3.
 9. An aqueouspharmaceutical composition having a pH of 5.0 to 7.0 comprising (i) ananti-CD40 antibody wherein the antibody has a concentration of at least50 mg/ml, and wherein said anti-CD40 antibody includes heavy chain CDR1,CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and light chainCDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8, (ii) a stabiliser, (iii)a buffering agent, (iv) a surfactant, and optionally (v) an amino acid.10. The aqueous pharmaceutical composition of claim 9 comprising (i) ananti-CD40 antibody wherein the antibody has a concentration of at least50 mg/ml, and wherein said anti-CD40 antibody includes heavy chain CDR1,CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and light chainCDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8, (ii) sucrose or trehaloseas a stabiliser, (iii) histidine as a buffering agent, (iv) polysorbate20 as a surfactant, and optionally (v) an amino acid selected fromarginine, methionine or glycine.
 11. The aqueous pharmaceuticalcomposition of claim 10 comprising (i) an anti-CD40 antibody wherein theantibody has a concentration of at least 50 mg/ml, and wherein saidanti-CD40 antibody includes heavy chain CDR1, CDR2 and CDR3 of SEQ IDNOs 3, 4 and 5 respectively, and light chain CDR1, CDR2 and CDR3 of SEQID NOs: 6, 7 and 8, (ii) 200-300 mM sucrose as a stabiliser, (iii) 25-35mM histidine as a buffering agent, (iv) up to 0.2% polysorbate 20 as asurfactant, and optionally (v) 10-80 mM arginine, methionine or glycine.12. The aqueous pharmaceutical composition of claim 10, wherein thecomposition has a pH of 6.0 and comprises (i) an anti-CD40 antibodywherein the antibody has a concentration of at least 50 mg/ml, andwherein said anti-CD40 antibody includes heavy chain CDR1, CDR2 and CDR3of SEQ ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2 andCDR3 of SEQ ID NOs: 6, 7 and 8, (ii) 270 mM sucrose as a stabiliser,(iii) 30 mM histidine as a buffering agent, and (iv) 0.06% polysorbate20 as a surfactant.
 13. The aqueous pharmaceutical composition of claim11, wherein the composition has a pH of 6.0 and comprises (i) ananti-CD40 antibody wherein the antibody has a concentration of at least50 mg/ml, and wherein said anti-CD40 antibody includes heavy chain CDR1,CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and light chainCDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8, (ii) 270 mM sucrose as astabiliser, (iii) 30 mM histidine as a buffering agent, (iv) 0.06%polysorbate 20 as a surfactant, and (v) 51 mM arginine.
 14. The aqueouspharmaceutical composition of claim 9, wherein the anti-CD40 antibodyhas a concentration of 150 mg/ml.
 15. The lyophilised formulation ofclaim 1, wherein the anti-CD40 antibody comprises a V_(H) domain havingthe amino acid sequence of SEQ ID NO: 1 and a V_(L) domain having theamino acid sequence of SEQ ID NO:
 2. 16. The lyophilised formulation ofclaim 1, wherein the anti-CD40 antibody comprises a heavy chain regionof SEQ ID NO: 9 and a light chain region of SEQ ID NO:
 10. 17. Thelyophilised formulation of claim 1, wherein the anti-CD40 antibodycomprises a heavy chain region of SEQ ID NO: 13 and a light chain regionof SEQ ID NO:
 14. 18. The lyophilised formulation of claim 1, whereinthe anti-CD40 antibody comprises a heavy chain region of SEQ ID NO: 17and a light chain region of SEQ ID NO:
 18. 19. A delivery deviceincluding the aqueous pharmaceutical composition of claim
 9. 20. Apre-filled syringe including the aqueous pharmaceutical composition ofclaim
 9. 21. A method for delivering an anti-CD40 antibody to a mammal,comprising a step of administering to the mammal an aqueouspharmaceutical composition as claimed in claim
 9. 22. A method oftreating a disease or disorder that is mediated by CD40 comprisingadministering to a patient in need thereof a lyophilised formulation ofclaim
 1. 23. The method of claim 22, wherein the disease or disorderthat is mediated by CD40 is an autoimmune disease.
 24. The method ofclaim 22, wherein the disease or disorder is multiple sclerosis,Sjögren's syndrome, transplant rejection, graft-versus-host disease,rheumatoid arthritis, systemic lupus erythematosus, or Pemphigusvulgaris.
 25. The aqueous pharmaceutical composition of claim 9, whereinthe anti-CD40 antibody comprises a V_(H) domain having the amino acidsequence of SEQ ID NO: 1 and a V_(L) domain having the amino acidsequence of SEQ ID NO:
 2. 26. The aqueous pharmaceutical composition ofclaim 9, wherein the anti-CD40 antibody comprises a heavy chain regionof SEQ ID NO: 9 and a light chain region of SEQ ID NO:
 10. 27. Theaqueous pharmaceutical composition of claim 9, wherein the anti-CD40antibody comprises a heavy chain region of SEQ ID NO: 13 and a lightchain region of SEQ ID NO:
 14. 28. The aqueous pharmaceuticalcomposition of claim 9, wherein the anti-CD40 antibody comprises a heavychain region of SEQ ID NO: 17 and a light chain region of SEQ ID NO: 18.29. A method for delivering an anti-CD40 antibody to a mammal,comprising a step of administering to the mammal an aqueouspharmaceutical composition as claimed claim
 9. 30. A method of treatinga disease or disorder that is mediated by CD40 comprising administeringto a patient in need thereof an aqueous pharmaceutical compositionaccording to claim
 9. 31. The method according to claim 30, wherein thedisease or disorder is an autoimmune disease.
 32. The method accordingto claim 31, wherein the disease or disorder is multiple sclerosis,Sjögren's syndrome, transplant rejection, graft-versus-host disease,rheumatoid arthritis, systemic lupus erythematosus, or Pemphigusvulgaris.